Human lung organoids develop into adult airway-like structures directed by physico-chemical biomaterial properties
Introduction
Human lung organoid models facilitate the study of cell fate decisions during development and for modeling diseases such as cystic fibrosis and goblet cell metaplasia, and infections such as respiratory syncytial virus [[1], [2], [3], [4], [5], [6], [7], [8]]. We have previously demonstrated that human pluripotent stem cell (hPSC)-derived human lung organoids (HLOs) possess a complex tissue structure in vitro, which includes both the epithelium and supporting tissue (cartilage, smooth muscle, fibroblasts) [1,9].
Notably, in vitro HLO cultures reflect the fetal airway, with adult airway-like structures generated only after in vivo transplantation [9]. Maturation following in vivo transplantation of HLOs reflects observations with numerous other organoid and hPSC-based systems [1,[10], [11], [12], [13]],. While many other studies have shown successful transplantation of hPSC-derived tissues under the kidney capsule or other vascular sites within the murine host, HLOs required the assistance of a PLG microporous polymer scaffold to support engraftment and vascularization following transplant into the epididymal fat pad of immunocompromised mice. After 8 weeks, the transplanted HLO (tHLO) had airway-like structures that resembled native adult airways including proper cellular organization, epithelial cellular ratios and airway cell types. Airway-like structures were also surrounded by smooth muscle and possessed cartilage, as would be the case in the human airways [9]. However, these previous studies did not identify the polymer scaffolds design parameters that conferred an engraftment and growth advantage for HLOs.
In this report, we investigated the physico-chemical properties of microporous scaffolds that support HLO maturation into airway structures. Polymers have different degradation rates and may have distinct interactions with the host, so microporous scaffold support of transplanted HLO were tested using diverse materials including poly (lactide-co-glycolide) (PLG), polycaprolactone (PCL), and poly (ethylene glycol) (PEG). The interconnected pore size was varied, as well, through the initial scaffold fabrication and also through the degradation rate of the polymers. For these material platforms, we investigated airway maturation, immune response, as well as overall explant and airway size. Identifying the biomaterial design parameters that influence airway maturation and structure will enable the development of platforms that can direct the structure to better model airway homeostasis and disease environments.
Section snippets
Maintenance of hESCs, generation of HLOs, and seeding on scaffolds
H1 human embryonic stem cell (hESC) line (NIH registry #0043) and H9 (NIH registry #0062) was obtained from the WiCell Research Institute. H1 hESC line was used to derive all HLOs for these experiments except for Fig. 2 where H9 hESC and H9 GFP hESC lines were used to derive HLOs. H9 GFP hESC line was generated by infecting H9 hESCs with pLenti PGK GFP Puro virus generated from the plasmid purchased from AddGene (Cat#: 19,070) [14]. H9 GFP hESC clonal line was generated by puromycin selection
PLG, PEG and PCL have varying extents of HLO derived airway maturation
Microporous scaffolds with similar architectures and formed from either 75:25 (lactide:glycolide) PLG or 20% (w/v) 4-arm PEG-maleimide microporous scaffold were tested for their ability to support transplantation of foregut spheroids. Both scaffolds had pores ranging in size from 225 μm to 450 μm, and were cylinder shaped with a diameter of 5 mm diameter and a thickness of 2 mm. PLG is a degradable, hydrophobic polyester that will adsorb proteins while PEG scaffolds are non-degradable
Discussion
In this report, we have demonstrated that the type of material and degradation of the microporous scaffold can affect lung airway formation, airway size, and explant size derived from transplanted HLOs (Table 2). Previously, 75:25 PLG microporous scaffolds were used to transplant HLOs into the epididymal fat pad [9]. Since no maturation occurred when HLOs were placed into the kidney capsule or sewn in the omentum of an immunocompromised mouse, we hypothesized that the tHLOs needed a surface to
Data availability
The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study.
Acknowledgments
This research was supported by funding from NIH grant R21 EB024410, R21 AI47677, and R01 EB009910.
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